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PLoS One. 2011; 6(8): e23415.
Published online Aug 12, 2011. doi:  10.1371/journal.pone.0023415
PMCID: PMC3155540

Comparative Genomics of Multidrug Resistance-Encoding IncA/C Plasmids from Commensal and Pathogenic Escherichia coli from Multiple Animal Sources

Ulrich Dobrindt, Editor

Abstract

Incompatibility group A/C (IncA/C) plasmids have received recent attention for their broad host range and ability to confer resistance to multiple antimicrobial agents. Due to the potential spread of multidrug resistance (MDR) phenotypes from foodborne pathogens to human pathogens, the dissemination of these plasmids represents a public health risk. In this study, four animal-source IncA/C plasmids isolated from Escherichia coli were sequenced and analyzed, including isolates from commercial dairy cows, pigs and turkeys in the U.S. and Chile. These plasmids were initially selected because they either contained the floR and tetA genes encoding for florfenicol and tetracycline resistance, respectively, and/or the blaCMY-2 gene encoding for extended spectrum β-lactamase resistance. Overall, sequence analysis revealed that each of the four plasmids retained a remarkably stable and conserved backbone sequence, with differences observed primarily within their accessory regions, which presumably have evolved via horizontal gene transfer events involving multiple modules. Comparison of these plasmids with other available IncA/C plasmid sequences further defined the core and accessory elements of these plasmids in E. coli and Salmonella. Our results suggest that the blaCMY-2 plasmid lineage appears to have derived from an ancestral IncA/C plasmid type harboring floR-tetAR-strAB and Tn21-like accessory modules. Evidence is mounting that IncA/C plasmids are widespread among enteric bacteria of production animals and these emergent plasmids have flexibility in their acquisition of MDR-encoding modules, necessitating further study to understand the evolutionary mechanisms involved in their dissemination and stability in bacterial populations.

Introduction

The use of antimicrobial agents in agriculture has been scrutinized over the past two decades because of their potential detrimental effects on animal and human health. Although the administration of antibacterial agents is an effective means to control bacterial infections, the use of antibiotics in agriculture is not limited to disease treatment and control; they are also used to prevent disease and to promote growth. Such use is postulated to facilitate the emergence of multidrug resistant bacteria isolated from animal sources (e.g., non-typhoidal Salmonella spp., Escherichia coli and other food-borne pathogens), and the dissemination of their multidrug resistance (MDR)-encoding determinants to other susceptible bacteria through horizontal gene transfer. The dissemination of MDR via conjugative plasmids can potentially limit future therapeutic options for treating infections in animals and humans [1], [2], [3], [4], [5], [6], [7].

Horizontal transfer of individual or arrays of resistance genes occurs mainly through the acquisition of conjugative plasmids, integrons, and transposons in enteric bacteria. Bacterial plasmids are self-replicating, extrachromosomal replicons, and as such they are key agents of genetic change in microbial populations. Besides conferring resistance to antibiotics, naturally occurring plasmids promote the spread of a variety of traits, including resistance to mercury and other heavy metals, virulence, fitness, and the metabolism of unusual compounds [6], [7], [8], [9], [10], [11], [12]. In recent years, there has been growing interest in the study of plasmids belonging to the IncA/C incompatibility group, mainly because of their ability to confer resistance to a diverse group of antimicrobial agents and their broad host range. IncA/C plasmids have been identified in numerous bacterial species, including Aeromonas hydrophila [13], [14], Yersinia pestis [15], [16], Photobacterium damselae subsp. Piscicida [17], [18], Klebsiella pneumoniae [19], Vibrio cholera [20], [21], E. coli [22], A. salmonicida [1], and S. enterica [8], [22]. Analysis of the completed sequences of these plasmids has revealed that, with the exception of accessory components containing resistance-encoding elements, they were virtually identical to one another [14], [16], [22]. Among the genes identified within the IncA/C plasmid accessory regions are those encoding for resistance to tetracycline (tetA), chloramphenicol/florfenicol (floR), streptomycin/spectinomycin (aadA2), sulfonamides (sul1 and sul2), and extended-spectrum β-lactamases (blaCMY-2). In addition, the recent epidemic emergence of strains containing the blaNDM-1 metallo-beta-lactamase gene, which are resistant to all antibiotic options in humans, has been associated with the IncA/C plasmid [23].

We recently completed the sequence of an IncA/C plasmid from E. coli isolated from a dairy cow in Illinois. This plasmid, approximately 165 kb in size, shared strong similarities with IncA/C plasmids isolated from human-source Salmonella, suggesting recent movements of this plasmid type among a variety of enteric populations [22]. The widespread distribution of IncA/C plasmids among E. coli and Salmonella necessitates studying their genetic repertoire and similarities with plasmids from other bacterial populations in order to fully understand their emergence and evolution in these species. Therefore, the aim of this study was to analyze genetic differences in several IncA/C plasmids from E. coli recovered from differing production animal sources and geographical locations using comparative plasmid sequencing and analysis.

Results

Sequence overview

Four plasmids were sequenced in this study, including the resequencing of pAR060302, previously isolated from a florfenicol-resistant E. coli commensal isolate from a US dairy cow [22]. The remaining three plasmids sequenced were from a commensal E. coli strain from a dairy cow in Chile (pPG010208), an avian pathogenic E. coli strain from a turkey in the USA with colibacillosis (p199061_160), and a porcine enterotoxigenic E. coli strain from a pig in the USA with post-weaning diarrhea (pUMNK88_161). All were sequenced using high-throughput Roche 454 DNA sequencing. These plasmids were isolated from farms in different geographical locations in the U.S. and Chile (Table 1). Single contiguous sequences with at least 15-fold coverage were obtained for each plasmid sequenced using draft assembly and PCR-based gap closure. BLAST analysis of the completed nucleotide sequences confirmed that they all belonged to the IncA/C incompatibility group based upon analysis of the predicted replicon. The sequence of pAR060302 was identical to the previous sequence generated via Sanger sequencing [22]. The plasmids varied in size from 135 to 165 kb, and with the exception of the accessory regions (see below), their backbone sequences were highly conserved (>99% nucleotide sequence similarity) and syntenic. Of the predicted open reading frames, approximately 40% were of unknown function. The predicted proteins with known function were primarily associated with resistance to antibiotics and heavy metals, conjugative transfer, and replication (Table S1).

Table 1
General characteristics of the IncA/C plasmids sequenced in this study.

sul2-containing accessory region

As described above, the sequenced plasmids differed primarily in their accessory regions. These regions mainly included insertion sequences and transposases, class 1 integrons, antibiotic resistance determinants, and heavy metal detoxification proteins. Analysis of these regions revealed the presence of several accessory gene clusters implicated in resistance to multiple antimicrobial agents. One of these regions occurs between repA and a putative conjugative transfer region (designated Tra1), and is a 16-kb module containing floR-tetA-strAB-sul2, encoding resistance to phenicols, tetracyclines, aminoglycosides, and sulfonamides (Fig. 1). This sul2-containing region is identical in plasmids pAR060302, pUMNK88_161, and p199061_160, with the exception of a truncated strB gene in p199061_160. Also, located upstream of the floR gene were two copies of IS26, two ORFs encoding unknown functions, and an ISCR2 element [24]. This region was also present in pPG010208; however, the mph2 and mel genes encoding for macrolide resistance and an additional IS26 element are also located upstream of this region (Fig. 1).

Figure 1
Comparison of the sul2 regions of the plasmids sequenced in this study.

Conjugative transfer and blaCMY-2-containing regions

Within the backbone of all of the sequenced IncA/C plasmids are two putative conjugative transfer-associated regions designated Tra1 and Tra2. The Tra1 region consists of 22 ORFs, including 9 conserved hypothetical proteins, all located in a single gene cluster (Fig. 2). In blaCMY-2-containing plasmids, one or more copies of blaCMY-2 is inserted within the Tra1 region in different locations. Among our sequenced plasmids, all except pPG010208 contained a blaCMY-2 insertion within Tra1. pAR060302 and pUMNK88_161 contained the insertion downstream of traA. In addition to blaCMY-2, this accessory module contains genes with homology to the blc, sugE, and dsbC genes as previously described [22]. Also, the insertion sequence ISEcp1, belonging to the IS1380 family, exists upstream of the blaCMY-2 gene in all cases. This gene varies in size from 948 bp in the swine-source E. coli plasmid (pUMNK88_161) to 1,262 bp in the avian- and bovine-source E. coli isolates' plasmids (p199061_160 and pAR060302) (Fig. 2). In 199061_160, a region containing traLEKBV upstream of the insertion is absent.

Figure 2
Comparison of the conjugative transfer and the blaCMY-2 containing regions of the plasmids sequenced in this study.

Class 1 integron-containing accessory region

A third accessory module exists in most sequenced IncA/C plasmids, separating a cluster of hypothetical genes and the Tra2 region. This region typically contains a Tn21-like class 1 integron structure with multiple antibiotic resistance gene cassettes and mercury resistance genes. This structure is absent from pPG010208, but present in the three other plasmids from this study inserted in identical locations. In pAR060302 and p199061_160, identical class 1 integrons are present that contain aminoglycoside resistance genes, aadA and aacC; heat-shock chaperones groSEL; the qacEdelta1 and the sul1 genes; and mercury resistance genes merDBAPTR. The integron region is flanked by IS4321 elements similar to that previously described for Tn21 [25]. In pUMNK88_161, the Tn21-like structure is identical to that of plasmids pAR060302 and p199061_160, except that the gene cassette region in pUMNK88_161 contains cmlA, encoding resistance to chloramphenicol, and aadA2, encoding aminoglycoside resistance.

Transcriptional regulators

The recent influx of available genome sequences in the public database has improved the ability to effectively annotate possible functions to predicted proteins based on inferred sequence similarity. Previously, most of the predicted proteins of the IncA/C plasmid were hypothetical proteins. Upon re-annotation of these sequences, six predicted transcriptional regulators have been identified on the IncA/C plasmid backbone. These include proteins with similarity to families HU-beta, H-NS, Xre, LysR, and LuxR. In the case of the H-NS-like and HU-like proteins, these represent novel orthologs with only 81% sequence similarity (HU-beta) and 52% sequence similarity (H-NS) to their closest matches (Fig. 3). Also, these proteins do not share any significant similarity with the previously described plasmid-encoded H-NS proteins Pmr and Sfh from IncP-7 and IncH plasmids [26], [27].

Figure 3
Amino acid sequence alignment of the predicted H-NS- and HU-like proteins of IncA/C plasmids.

G+C content

The G+C contents of each plasmid sequenced here were analyzed and compared to the archetypic IncA/C plasmid pRA1 isolated in 1971 from the fish pathogen Aeromonas hydrophila (Figure S1). Local G+C content varied from approximately 28% to 73% across the different plasmids, with two regions of high G+C content observed (~60%). The first corresponded to the accessory module containing the floR, tetA and sul2 genes, which is absent in pRA1. The other high G+C content fragment corresponds to the Tn21-like accessory regions, present on p199061_160, pUMNK88_161, and pAR060302. Additionally, two low G+C content regions were observed (~30%) corresponding to the genes that confer resistance to macrolides and the conjugative transfer Tra1 region containing blaCMY-2. These regions of low or high G+C content were generally flanked by IS elements or inverted repeats.

Comparison of all sequenced IncA/C plasmids

Linear maps were constructed for twelve completed IncA/C plasmids, including those from this study and from A. hydrophila (pRA1), Y. ruckeri (pYR1), P. damselae (pP91278 and pP99-018), Y. pestis (pIP1202), E. coli (peH4H), and S. enterica (pAM04528) (Figs. 4 and and5)5) [22]. As previously determined, the core backbones of these plasmids are all highly syntenic with no genetic rearrangements [14], [22]. The plasmids were grouped into those lacking (Fig. 4) or possessing (Fig. 5) the blaCMY-2 insertion. Eleven of twelve plasmids had sul2 in an accessory region between repA and Tra1. All of the blaCMY-2 plasmids had an identical sul2 accessory region structure containing floR-tetAR-strBA-sul2. The sul2 regions from plasmids pPG010208, pIP1202, pP99-018, pP91278, and pYR1 all differed from the blaCMY-2 plasmids and each other. The blaCMY-2 insertions within Tra1 varied with regard to insertion location, copy number, and genetic layout. p199061_160, pUMNK88_161, and pAR060302 all contained a single blaCMY-2 insertion downstream of traA, with the deletion of several Tra1 genes from p199061_160. peH4H contained duplicate blaCMY-2 insertions in Tra1 with multiple truncations of the Tra1 region. pAM04528 and pSN254 both contained adjacent and inverted copies of the blaCMY-2 region downstream of traA.

Figure 4
Linear maps of sequenced IncA/C plasmids lacking blaCMY-2.
Figure 5
Linear maps of sequenced IncA/C plasmids possessing blaCMY-2.

The third accessory region site lies upstream of the Tra2 region, and generally involves integron-like elements (Figures 4 and and5).5). In the blaCMY-2 plasmids, all of these insertions involve intact Tn21 or remnants thereof, inserted into identical sites. All of these regions contain a mercury resistance operon. In all blaCMY-2 plasmids except pAM04528, a class 1 integron is upstream of mer and varies between plasmids only with regard to gene cassette content. In pAM04528, mer is present but the class 1 integron appears to have been deleted. Among the non-blaCMY-2 plasmids, three (pP91278, pP99-018, and pPG010208) do not have an accessory element upstream of Tra2. One plasmid, pIP1202, contains a Tn21-like structure like the blaCMY-2 plasmids except that it is inverted. pRA1 and pYR1 contain insertions upstream of Tra2 that do not involve Tn21. In pRA1, a sul2 element with tetAR is present, and in pYR1 Tn10 and strAB are present.

Discussion

It is increasingly evident that IncA/C plasmids have emerged among populations of human and animal enteric bacteria, particularly E. coli, Salmonella, and Klebsiella spp. To better understand the genetic structure of these plasmids in production animal E. coli populations, we sequenced three plasmids from E. coli that had been isolated from a healthy commercial dairy cow, a diseased pig, and a diseased turkey, and re-sequenced a fourth plasmid from a commercial dairy cow. These strains and plasmids were selected for their ability to confer resistance to florfenicol. DNA sequencing confirmed that all four plasmids belong to the IncA/C group. Plasmids from this group have been isolated and described previously from a variety of Proteobacteria from animals, soil, and water. These include both pathogens and commensal bacteria [8], [14], [22], [23], [28]. Despite the fact that the IncA/C plasmids sequenced thus far were isolated from different geographical locations and diverse sources, the growing collection of IncA/C plasmid sequences all share a remarkably conserved backbone with varying accessory elements collectively encoding for a broad spectrum of antimicrobial resistance. The E. coli- and Salmonella-source IncA/C plasmids sequenced thus far are quite similar in that they all have similar accessory regions, including the sul2-containing, blaCMY-2-containing (except pPG010208), and class 1 integron-containing modules. Temporal screening of historical Salmonella Newport and E. coli from humans and animals dating from 1940 to date suggests that IncA/C plasmids emerged after 1980 [29], [30], which is suggestive that a transfer event might have occurred prior to this time resulting in the introduction of a prototype IncA/C plasmid into these populations. However, plasmid and resistance phenotypes of IncA/C plasmids in recently isolated E. coli and Salmonella suggest that a variety of IncA/C plasmid variants exist in these populations with differing resistance phenotypes and genetic content [31], [32], [33], [34]. Therefore, it is unclear is these variants have arisen from recombinational events while in these species, or if multiple plasmid introductions have occurred.

The comparison of IncA/C plasmids from different production animals and geographic locations provides further evidence that the blaCMY-2 plasmids represent a unique IncA/C lineage that appears to be quite successful among bacterial populations, since they have been increasingly isolated and identified [35], [36], [37], [38], [39], [40], [41], [42]. Analyses of this lineage suggests that its basic structure, in addition to the IncA/C conserved components among all sequenced plasmids, also includes a sul2 module containing floR-tetAR-strAB and a Tn21-like module. However, while the sul2 module appears to be stably maintained among this lineage, the blaCMY-2 and Tn21-like regions appear to be in constant flux. The ISEcp1 element is associated with all copies of IncA/C-encoded blaCMY-2. ISEcp1, like ISCR elements, is involved in ‘one-ended transposition’ and has the ability to mobilize itself and adjacent resistance-associated genes [43]. Mobilization and duplication of beta-lactamase genes mediated by ISEcp1-like elements are well described in multiple bacterial species [44]. This helps to explain the variable duplication of the blaCMY-2-ISEcp1 module throughout the Tra1 region on IncA/C plasmids. The floR and sul2 genes have been associated with ISCR2 in numerous other genetic contexts [24], suggesting that ISCR2 was involved in the introduction of the floR-tetA-strAB-sul2 element that is conserved in this lineage of IncA/C plasmids. In the class 1 integron region, most sequenced plasmids contain an ISCR16 element adjacent to the groESL genes, as previously described [24], [45], [46]. Overall, the blaCMY-2-containing IncA/C plasmids are remarkable in that they contain at least three ‘integration hotspots’ for the acquisition of accessory genetic modules; they contain multiple means of acquiring these elements, including gene cassette acquisition via integrons, classical IS-mediated acquisition via IS26 elements; and ‘one-ended’ acquisition via ISEcp1 and ISCR elements; and are of apparent broad host range [47].

The backbone of the IncA/C plasmid contains a number of putative DNA binding transcriptional regulators classified as nucleoid-associated proteins (NAPs). Such proteins are named for their ability to fold chromosomal DNA and form the nucleoid within the bacterial cell, and are well studied and also known for their immense regulatory properties. NAPs are categorized into several groups, including Fis, H-NS, HU, IHF, and Lrp [48]. H-NS is known to bind to A+T-rich regions and acts as a global transcriptional repressor; HU is also a global regulator that binds to DNA non-specifically. While the most-studied NAPs are those encoded on the bacterial chromosome, a number of plasmids have also been shown to possess NAPs. The first plasmid type identified with an H-NS NAP homolog, Sfr, was the IncH plasmid R27 from S. enterica serovar Typhimurium [49]. The effects of this plasmid and its Δsfh mutant were studied in S. Typhimurium. Interestingly, when pR27 was introduced into S. Typhimurium a limited number of chromosomal genes were differentially expressed, but the introduction of pR27Δsfh resulted in a nearly 4-fold increase in the number of chromosomal genes affected [49]. Furthermore, the Δsfh mutation greatly increased the fitness cost of carrying pR27 to the bacterial host. These observations were termed “stealth functions” elicited by such plasmid-encoded NAPs for their ability to silence the effects of pR27 on the host chromosome. Follow-up chromatin immunoprecipitation (ChIP) studies found that Sfh acts to bind to regions within the H-NS regulatory network and thus minimizes the effects of pR27 acquisition on the host chromosome regulatory networks [27]. A second plasmid-encoded H-NS-like protein with stealth function (Pmr) was identified on IncP-7 plasmids [26]. It is possible that the nucleoid-associated proteins encoded on IncA/C plasmids could elicit similar and immense effects on the transcriptional regulatory networks of their hosts, resulting in decreases in fitness costs and increases in host range associated with this plasmid group. However, the H-NS- and HU-like proteins identified on IncA/C plasmids are novel and only share low amino acid sequence similarity with their closest matches (Fig. 3); therefore, the roles of these proteins in such activities would need to be experimentally characterized. Certainly, further studies involving the biological mechanisms by which IncA/C plasmids succeed in various hosts are warranted, given their immense dissemination and association with pan-resistance.

A potential predecessor to the blaCMY-2 lineage of IncA/C plasmids is hinted at by the sequence of pPG010208 from a Chilean bovine-source E. coli isolate, which contains the sul2 region identical to blaCMY-2 plasmids but lacks blaCMY-2 itself and lacks a Tn21-like accessory region. This plasmid has additionally acquired the mel and mph-2 genes surrounded by two IS26 copies upstream of ISCR2, present only on pPG010208 as compared to other sequenced plasmids. These genes confer resistance to macrolides, which are mainly active against Gram-positive bacteria and are considered the drug of choice for group A streptococcal and pneumococcal infections when penicillin cannot be used [50]. Similar genetic structures to this have been described on plasmid pMUR050, isolated from an E. coli strain from a diarrheagenic pig [51] and on the pCTX-M3, a highly conjugative plasmid responsible for the dissemination of blaCTX genes in clinical populations of the family Enterobacteriaceae in Poland [52]. The differences observed between the Chilean isolate plasmid and other sequenced plasmids from U.S. isolates could represent an “isolation by distance” scenario, where differing local pressures could affect the acquisition of accessory elements in these plasmids. Ceftiofur is used frequently in the dairy industry of Chile, including on the farm where this isolate was obtained, but in our experience resistance to third generation cephalosporins and the blaCMY-2 gene encoding this ability are rarely identified among Chilean E. coli isolates. The discrepancies observed between pPG010208 and other sequenced IncA/C plasmids are not fully understood from an evolutionary and selective pressure standpoint, and deserve further study.

In addition to the accessory elements, we detected differences on the conjugal transfer system of the sequenced plasmids. In the non-blaCMY-2 plasmids, their Tra1 and Tra2 regions were generally complete and intact. However, the mosaic nature of the blaCMY-2 insertions and their duplications within the Tra1 region resulted in apparent disruptions of this region. For example, p199061_160 lacks of a 4-kb segment that includes the traEKBVA genes, which is present in pUMNK88_161 and pAR060302. Also, the blaCMY-2 insertion in several of these plasmids disrupts the traA and traC genes. Poole et al. studied the conjugative transferability of IncA/C plasmids containing or lacking the blaCMY-2 gene in Salmonella, concluding that plasmids encoding blaCMY-2 were rarely transferred compared with higher conjugation efficiencies where blaCMY-2 was absent [37]. Call et al. also reported the failure of self-conjugation for some of the IncA/C plasmids [22]. They reported that the failure of transferability of some of the IncA/C plasmids in their study was due to differences of the tra genes localized with blaCMY-2. Others have noted that blaCMY-2 insertions do not necessarily affect the conjugative ability of IncA/C plasmids [31]. Thus, possible transfer deficiencies conferred through blaCMY-2 acquisition, the role of co-residing plasmids in decreasing its fitness cost and increasing its conjugative frequency, and dissection of the Tra1 and Tra2 regions in conjugative transfer still need to be studied.

A key question pertaining to multidrug resistance encoded by IncA/C plasmids is their maintenance in bacterial populations in the absence of selective pressures. Third-generation cephalosporins such as ceftriaxone and ceftiofur have important applications to both human and animal health [53]. Various genes encode for proteins that confer reduced susceptibility to these antimicrobials, and blaCMY-2 is commonly responsible for the resistance to these antimicrobial agents in the U.S. [22], [35], [37], [39], [54], [55]. Potential risks have been identified due to the possible co-selection of the blaCMY-2 through the use of florfenicol in food animal production. Compounding this scenario is the presence of multiple means of selection, including antibiotics and heavy metals, on many IncA/C plasmids. It was recently demonstrated the long-term maintenance of IncA/C plasmids might require selective pressure, which contrasts the apparent success of this plasmid type in a variety of environmental niches including possible non-selective environments [56]. This underscores the need to elucidate the selective pressures that drive the success of this plasmid type in Enterobacteriaceae.

The presence of the cmlA gene on the class 1 integrons was another trait that differed between the IncA/C plasmid sequences. This gene was detected only on the swine-source E. coli plasmid, pUMNK88_161 (Figure 1). These results agree with the study by Bischoff et al., who found that 48 of the 90 E. coli isolates from swine production in Oklahoma exhibited resistance to chloramphenicol and 47 of these isolates possessed the cmlA gene [41]. This gene encodes a putative efflux pump that confers resistance to chloramphenicol, which has been banned in the U.S. since 1985. Thus, the presence of this gene on IncA/C plasmids is an example of an additional selection mechanisms for its widespread dissemination in commercial pig hosts and persistence in the absence of the particular selective pressures, and is aggravated by the fact that the use of any antimicrobial encoded by the IncA/C plasmid can potentially co-select for a number of additional phenotypes.

Previous studies of the IncA/C plasmids suggest that these plasmids probably did share a recent common ancestor. Fricke et al. sequenced pRA1 [14], considered the first member of the IncA/C group of MDR plasmids to be fully described [57]. This plasmid showed a reduced antimicrobial resistance spectrum, which the authors attributed to a probable minimal selective pressure. The authors proposed an evolutionary model in which each “IncA/C plasmid diverged from a common ancestor through a specific process of stepwise integration events of horizontally acquired resistance gene arrays” [14]. It appears that the blaCMY-2 plasmid lineage is such an example, where its emergence resulted from initial acquisitions of its sul2 module, blaCMY-2 module, and Tn21. Further evolution of this plasmid lineage and other IncA/C lineages seems to be rapidly occurring, as recent reports have identified the New Delhi Metallo-β-Lactamase (NDM-1) occurring on or with IncA/C plasmids [23], [58]. Again, the underlying mechanisms driving the evolution and emergence of such IncA/C plasmid variants is unclear, but will likely present great challenges to human and animal health.

In conclusion, variants of broad-host-range IncA/C plasmids have emerged in a variety of bacterial species. The association of MDR with integrons, complex transposons, and ISCR elements, all on a conjugative plasmid, infers the possibility of dissemination among clinical isolates that creates opportunities for the rapid emergence of multidrug resistant bacterial clones. Strains harboring these plasmids serve as a reservoir for antibiotic resistance genes, the further spread of which could likely limit therapeutic options. Based upon the recent analyses of IncA/C plasmids revealing their genetic components and dissemination among E. coli and Salmonella of humans and production animals, future studies are essential to determine the specific mechanisms of acquisition, persistence, and dissemination of these plasmids among bacterial populations.

Materials and Methods

Bacterial isolates

All strains used in this study are listed in Table 1. Isolates were collected in previous studies by the investigators [9], [22]. The research in these studies complied with all relevant animal use federal guidelines and institutional policies. The strains were selected because they all harbored a large plasmid and exhibited resistance to ceftriaxone, florfenicol or tetracycline. All strains were cultured at 37°C in Tryptone soy agar (TSA) and stored in 40% glycerol at −80°C.

Plasmid isolation and sequencing

Single colonies were inoculated into 100 mL LB broth and grown overnight at 37°C with shaking. Plasmid isolation was performed using Plasmid Midi Kit (Qiagen Inc., Valencia, CA). After purification, plasmid DNA was resuspended in sterile water and detected by electrophoresis on 0.8% agarose gels at 4°C. Ten micrograms of purified plasmid DNA was sequenced at Biomedical Genomic Center at the University of Minnesota using the Roche 454 GS-Titanium sequencing platform (454 Life Sciences, Branford, CT).

Assembling and annotation

For each strain, sequencing reads were assembled de novo using SeqMan software from DNAStar (Lasergene, Madison, WI). Assembled contigs were then mapped to a reference genome (FJ621588) for arrangement of the contiguous sequences in their most likely orientation. The final gaps were closed using standard PCR. Open Reading Frames (ORFs) in the plasmids sequences were identified using GeneQuest from DNAStar (Lasergene, Madison, WI), and ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/), followed by complete manual inspection. Translated ORFs were then compared to known protein sequences using BLAST [59]. Those with greater than 80% homology with database protein sequences were considered matches, while hypothetical proteins with greater than 80% amino acid sequence identity to one or more previously published proteins were classified as conserved hypothetical proteins. Insertion sequences and repetitive elements were identified using IS FINDER (http://www-is.biotoul.fr/). Finished sequences are deposited in Genbank under accession numbers HQ023864 (pAR060302), HQ023861 (pPG010208), HQ023862 (pUMNK88_161), and HQ023863 (p199061_160).

Comparative genomics

Following annotation, the assembled nucleotide sequences were analyzed to other plasmid sequences using BlastN [60]. Through this analysis, comparative linear maps (http://www.iayork.com/XPlasMap/) were created. The G+C content of each plasmid was analyzed using ARTEMIS software [61].

Antimicrobial susceptibility testing

Wild type strains were subjected to disk diffusion to determine the susceptibilities of isolates to the following drugs: streptomycin, tetracycline, florfenicol, chloramphenicol, ampicillin, and sulfisoxazole.

Supporting Information

Figure S1

Sliding G+C contents of each plasmid sequenced in this study. A = region containing floR, tetA and sul2 genes, B = the Tn21-like accessory regions, C = genes that confer resistance to macrolides, and D = the conjugative transfer region together with blaCMY-2 gene.

(TIF)

Table S1

Annotated features of sequenced plasmids.

(XLS)

Acknowledgments

The authors wish to thank the University of Minnesota Veterinary Diagnostic Laboratory and Lisa K. Nolan, Iowa State University, for providing strains for this study. This work was carried out in part using computing resources at the University of Minnesota Supercomputing Institute.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This project was supported in part by USDA NRICGP Grants 2000-35212-9398 and 2003-35212-13853 (Singer), NSF Grant 0405419 (Singer), and the Minnesota Pork Board (Johnson). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.

References

1. McIntosh D, Cunningham M, Ji B, Fekete FA, Parry EM, et al. Transferable, multiple antibiotic and mercury resistance in Atlantic Canadian isolates of Aeromonas salmonicida subsp. salmonicida is associated with carriage of an IncA/C plasmid similar to the Salmonella enterica plasmid pSN254. J Antimicrob Chemother. 2008;61:1221–1228. [PMC free article] [PubMed]
2. Norman A, Hansen LH, She QX, Sorensen SJ. Nucleotide sequence of pOLA52: A conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux. Plasmid. 2008;60:59–74. [PubMed]
3. Paterson DL. Resistance in gram-negative bacteria: Enterobacteriaceae. American Journal of Infection Control. 2006;34:S20–S28. [PubMed]
4. Hammerum AM, Heuer OE. Human health hazards from antimicrobial-resistant Escherichia coli of animal origin. Clini Infect Dis. 2009;48:916–921. [PubMed]
5. Johnson TJ, Skyberg J, Nolan LK. Multiple antimicrobial resistance region of a putative virulence plasmid from an Escherichia coli isolate incriminated in avian colibacillosis. Avian Dis. 2004;48:351–360. [PubMed]
6. Fricke WF, McDermott PF, Mammel MK, Zhao SH, Johnson TJ, et al. Antimicrobial Resistance-Conferring Plasmids with similarity to virulence plasmids from avian pathogenic Escherichia coli strains in Salmonella enterica serovar Kentucky isolates from poultry. Appl Environmen Microbiol. 2009;75:5963–5971. [PMC free article] [PubMed]
7. Johnson TJ, Jordan D, Kariyawasam S, Stell AL, Bell NP, et al. Sequence analysis and characterization of a transferable hybrid plasmid encoding multidrug resistance and enabling zoonotic potential for extraintestinal Escherichia coli. Infect Immun. 2010;78:1931–1942. [PMC free article] [PubMed]
8. Lindsey RL, Fedorka-Cray PJ, Frye JG, Meinersmann RJ. IncA/C plasmids are prevalent in multidrug-resistant Salmonella enterica isolates. Appl Environ Microbiol. 2009;75:1908–1915. [PMC free article] [PubMed]
9. Johnson TJ, Nolan LK. Plasmid replicon typing. Methods Mol Biol. 2009;551:27–35. [PubMed]
10. Frost LS, Leplae R, Summers AO, Toussaint A. Mobile genetic elements: the agents of open source evolution. Nat Rev Microbiol. 2005;3:722–732. [PubMed]
11. Shintani M, Takahashi Y, Tokumaru H, Kadota K, Hara H, et al. Response of the Pseudomonas host chromosomal transcriptome to carriage of the IncP-7 plasmid pCAR1. Environmen Microbiol. 2010;12:1413–1426. [PubMed]
12. DebRoy C, Sidhu MS, Sarker U, Jayarao BM, Stell AL, et al. Complete sequence of pEC14_114, a highly conserved IncFIB/FIIA plasmid associated with uropathogenic Escherichia coli cystitis strains. Plasmid. 2010;63:53–60. [PubMed]
13. Aoki T, Egusa S, Ogata Y, Watanabe T. Detection of resistance factors in fish pathogen Aeromonas liquefaciens. J Gen Microbiol. 1971;65:343–349. [PubMed]
14. Fricke WF, Welch TJ, McDermott PF, Mammel MK, LeClerc JE, et al. Comparative genomics of the IncA/C multidrug resistance plasmid family. J Bacteriol. 2009;191:4750–4757. [PMC free article] [PubMed]
15. Galimand M, Guiyoule A, Gerbaud G, Rasoamanana B, Chanteau S, et al. Multidrug resistance in Yersinia pestis mediated by a transferrable plasmid. N Engl J Med. 1997;337:677–680. [PubMed]
16. Welch TJ, Fricke WF, McDermott PF, White DG, Rosso ML, et al. Multiple antimicrobial resistance in plague: an emerging public health risk. PLoS One. 2007;2:e309. [PMC free article] [PubMed]
17. Kim E, Aoki T. Sequence analysis of the florfenicol resistance gene encoded in the transferable R-plasmid of a fish pathogen, Pasteurella piscicida. Microbiol Immunol. 1996;40:665–669. [PubMed]
18. Kim MJ, Hirono I, Kurokawa K, Maki T, Hawke J, et al. Complete DNA sequence and analysis of the transferable multiple-drug resistance plasmids (R Plasmids) from Photobacterium damselae subsp. piscicida isolates collected in Japan and the United States. Antimicrob Agents Chemother. 2008;52:606–611. [PMC free article] [PubMed]
19. Cloeckaert A, Baucheron S, Chaslus-Dancla E. Nonenzymatic chloramphenicol resistance mediated by IncC plasmid R55 is encoded by a floR gene variant. Antimicrob Agents Chemother. 2001;45:2381–2382. [PMC free article] [PubMed]
20. Hochhut B, Lotfi Y, Mazel D, Faruque SM, Woodgate R, et al. Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae O139 and O1SXT constins. Antimicrob Agents Chemother. 2001;45:2991–3000. [PMC free article] [PubMed]
21. Pan JC, Ye R, Wang HQ, Xiang HQ, Zhang W, et al. Vibrio cholerae O139 multiple-drug resistance mediated by Yersinia pestis pIP1202-like conjugative plasmids. Antimicrob Agents Chemother. 2008;52:3829–3836. [PMC free article] [PubMed]
22. Call DR, Singer RS, Meng D, Broschat SL, Orfe LH, et al. blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids. Antimicrob Agents Chemother. 2010;54:590–596. [PMC free article] [PubMed]
23. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis. 2010;10:597–602. [PMC free article] [PubMed]
24. Toleman MA, Bennett PM, Walsh TR. ISCR elements: novel gene-capturing systems of the 21st century? Microbiol Mol Biol Rev. 2006;70:296–316. [PMC free article] [PubMed]
25. Liebert CA, Hall RM, Summers AO. Transposon Tn21, flagship of the floating genome. Microbiol Mol Biol Rev. 1999;63:507–522. [PMC free article] [PubMed]
26. Yun CS, Suzuki C, Naito K, Takeda T, Takahashi Y, et al. Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function. J Bacteriol. 2010;192:4720–4731. [PMC free article] [PubMed]
27. Dillon SC, Cameron AD, Hokamp K, Lucchini S, Hinton JC, et al. Genome-wide analysis of the H-NS and Sfh regulatory networks in Salmonella Typhimurium identifies a plasmid-encoded transcription silencing mechanism. Mol Microbiol. 2010;76:1250–1265. [PubMed]
28. Welch TJ, Evenhuis J, White DG, McDermott PF, Harbottle H, et al. IncA/C plasmid-mediated florfenicol resistance in the catfish pathogen Edwardsiella ictaluri. Antimicrob Agents Chemother. 2009;53:845–846. [PMC free article] [PubMed]
29. Singh A, Zhao S, Sabo JL, Abbott JW, Fields PI, et al. 2011. Plasmid replicon typing of historical Salmonella Newport: 1940–2000; New Orleans, LA.
30. Tadesse DA, Zhao S, Harbottle H, Mcdermott PF. 2011. Plasmid diversity among historical E. coli isolates of human and animal origin, 1950s–2000s; New Orleans, LA.
31. Wiesner M, Calva E, Fernandez-Mora M, Cevallos MA, Campos F, et al. Salmonella Typhimurium ST213 is associated with two types of IncA/C plasmids carrying multiple resistance determinants. BMC Microbiol. 2011;11:9. [PMC free article] [PubMed]
32. Glenn LM, Lindsey RL, Frank JF, Meinersmann RJ, Englen MD, et al. Analysis of antimicrobial resistance genes detected in multidrug-resistant Salmonella enterica serovar Typhimurium isolated from food animals. Microb Drug Resist., Epub Ahead of Print 2011 [PubMed]
33. Lindsey RL, Frye JG, Thitaram SN, Meinersmann RJ, Fedorka-Cray PJ, et al. Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis. Microb Drug Resist. 2011;17:157–163. [PubMed]
34. Lindsey RL, Fedorka-Cray PJ, Frye JG, Meinersmann RJ. IncA/C plasmids are prevalent in multidrug-resistant Salmonella enterica isolates. Appl Environ Microbiol. 2009;75:1908–1915. [PMC free article] [PubMed]
35. Alcaine SD, Sukhnanand SS, Warnick LD, Su WL, McGann P, et al. Ceftiofur-resistant Salmonella strains isolated from dairy farms represent multiple widely distributed subtypes that evolved by independent horizontal gene transfer. Antimicrobial Agents and Chemotherapy. 2005;49:4061–4067. [PMC free article] [PubMed]
36. Brinas L, Moreno MA, Zarazaga M, Porrero C, Saenz Y, et al. Detection of CMY-2, CTX-M-14, and SHV-12 beta-lactamases in Escherichia coli fecal-sample isolates from healthy chickens. Antimicrob Agents Chemother. 2003;47:2056–2058. [PMC free article] [PubMed]
37. Carattoli A, Tosini F, Giles WP, Rupp ME, Hinrichs SH, et al. Characterization of plasmids carrying CMY-2 from expanded-spectrum cephalosporin-resistant Salmonella strains isolated in the United States between 1996 and 1998. Antimicrob Agents Chemother. 2002;46:1269–1272. [PMC free article] [PubMed]
38. Dierikx C, van Essen-Zandbergen A, Veldman K, Smith H, Mevius D. Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry. Vet Microbiol. 2010;145:273–278. [PubMed]
39. Doublet B, Carattoli A, Whichard JM, White DG, Baucheron S, et al. Plasmid-mediated florfenicol and ceftriaxone resistance encoded by the floR and bla(CMY-2) genes in Salmonella enterica serovars Typhimurium and Newport isolated in the United States. FEMS Microbiol Lett. 2004;233:301–305. [PubMed]
40. Frye JG, Fedorka-Cray PJ. Prevalence, distribution and characterisation of ceftiofur resistance in Salmonella enterica isolated from animals in the USA from 1999 to 2003. Int J Antimicrob Agents. 2007;30:134–142. [PubMed]
41. Gonzalez-Sanz R, Herrera-Leon S, de la Fuente M, Arroyo M, Echeita MA. Emergence of extended-spectrum beta-lactamases and AmpC-type beta-lactamases in human Salmonella isolated in Spain from 2001 to 2005. J Antimicrob Chemother. 2009;64:1181–1186. [PubMed]
42. Zaidi MB, Leon V, Canche C, Perez C, Zhao S, et al. Rapid and widespread dissemination of multidrug-resistant blaCMY-2 Salmonella Typhimurium in Mexico. J Antimicrob Chemother. 2007;60:398–401. [PubMed]
43. Toleman MA, Walsh TR. Combinatorial events of insertion sequences and ICE in Gram-negative bacteria. FEMS Microbiol Rev., Epub Ahead of Print 2011 [PubMed]
44. D'Andrea MM, Literacka E, Zioga A, Giani T, Baraniak A, et al. Evolution and spread of a multidrug-resistant Proteus mirabilis clone with chromosomal AmpC-type cephalosporinases in Europe. Antimicrob Agents Chemother. 2011;55:2735–2742. [PMC free article] [PubMed]
45. Toleman MA, Walsh TR. Evolution of the ISCR3 group of ISCR elements. Antimicrob Agents Chemother. 2008;52:3789–3791. [PMC free article] [PubMed]
46. Toleman MA, Walsh TR. ISCR elements are key players in IncA/C plasmid evolution. Antimicrob Agents Chemother. 2010;54:3534; author reply 3534. [PMC free article] [PubMed]
47. Suzuki H, Yano H, Brown CJ, Top EM. Predicting plasmid promiscuity based on genomic signature. J Bacteriol. 2010;192:6045–6055. [PMC free article] [PubMed]
48. Dorman CJ. Nucleoid-associated proteins and bacterial physiology. Adv Appl Microbiol. 2009;67:47–64. [PubMed]
49. Doyle M, Fookes M, Ivens A, Mangan MW, Wain J, et al. An H-NS-like stealth protein aids horizontal DNA transmission in bacteria. Science. 2007;315:251–252. [PubMed]
50. Noguchi N, Takada K, Katayama J, Emura A, Sasatsu M. Regulation of transcription of the mph(A) gene for macrolide 2′-phosphotransferase I in Escherichia coli: characterization of the regulatory gene mphR(A). J Bacteriol. 2000;182:5052–5058. [PMC free article] [PubMed]
51. Gonzalez-Zorn B, Teshager T, Casas M, Porrero MC, Moreno MA, et al. armA and aminoglycoside resistance in Escherichia coli. Emerg Infect Dis. 2005;11:954–956. [PMC free article] [PubMed]
52. Golebiewski M, Kern-Zdanowicz I, Zienkiewicz M, Adamczyk M, Zylinska J, et al. Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum beta-lactamase gene blaCTX-M-3. Antimicrob Agents Chemother. 2007;51:3789–3795. [PMC free article] [PubMed]
53. Singer RS, Patterson SK, Wallace RL. Effects of therapeutic ceftiofur administration to dairy cattle on Escherichia coli dynamics in the intestinal tract. Appl Environ Microbiol. 2008;74:6956–6962. [PMC free article] [PubMed]
54. Folster JP, Pecic G, Bolcen S, Theobald L, Hise K, et al. Characterization of extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg isolated from humans in the United States. Foodborne Pathog Dis. 2010;7:181–187. [PubMed]
55. Zhao S, White DG, Friedman SL, Glenn A, Blickenstaff K, et al. Antimicrobial resistance in Salmonella enterica serovar Heidelberg isolates from retail meats, including poultry, from 2002 to 2006. Appl Environ Microbiol. 2008;74:6656–6662. [PMC free article] [PubMed]
56. Subbiah M, Top EM, Shah DH, Call DR. Selection pressure required for long-term persistence of blaCMY-2-positive IncA/C plasmids. Appl Environ Microbiol. 2011;77:4486–4493. [PMC free article] [PubMed]
57. Bradley DE. Conjugation system of IncC plasmid RA1, and the interaction of RA1 pili with specific RNA phage C-1. Res Microbiol. 1989;140:439–446. [PubMed]
58. Poirel L, Schrenzel J, Cherkaoui A, Bernabeu S, Renzi G, et al. Molecular analysis of NDM-1-producing enterobacterial isolates from Geneva, Switzerland. J Antimicrob Chemother 2011 [PubMed]
59. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research. 1997;25:3389–3402. [PMC free article] [PubMed]
60. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7:203–214. [PubMed]
61. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, et al. Artemis: sequence visualization and annotation. Bioinformatics. 2000;16:944–945. [PubMed]
62. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular biology and evolution. 2007;24:1596–1599. [PubMed]

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